SPEN (spen family transcription repressor) is a nucleic acid-binding protein putatively involved in repression of gene expression. We hypothesized that SPEN could be involved in general downregulation of the transcription during the heat shock response in mouse spermatogenic cells through its interactions with chromatin. We documented predominant nuclear localization of the SPEN protein in spermatocytes and round spermatids, which was retained after heat shock. Moreover, the protein was excluded from the highly condensed chromatin. Chromatin immunoprecipitation experiments clearly indicated interactions of SPEN with chromatinin vivo. However, ChIP-Seq analyses did not reveal any strong specific peaks both in untreated and heat shocked cells, which might suggest dispersed localization of SPEN and/or its indirect binding to DNA. Usingin situproximity ligation assay we found closein vivoassociations of SPEN with MTA1 (metastasis-associated 1), a member of the nucleosome remodeling complex with histone deacetylase activity, which might contribute to interactions of SPEN with chromatin.